MUTATIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS TYPE-16 E6 PROTEIN - TRANSFORMING FUNCTION FOR HUMAN-CELLS AND DEGRADATION OF P53 IN-VITRO

The E6 oncoprotein of human papillomavirus type 16(HPV 16) [151 amino acids (AA) long] contains four metal-binding motifs, C-X-X-C, and is postulated to form two 29-AA finger-like structures in the N-terminal and C-terminal halves, which mediate degradation of p53 and binding to p53, respectively. We constructed a series of E6 mutants with single-AA substitutions in these finger regions (AAs 34-62 and 107-135) and examined their transforming function for human embryonic kidney (HEK) cells in conjunction with HPV 16 E7 and their interaction with human p53 in vitro. The mutants with substitution of L for F-37, G for L-50, S for Y-54, and P for L-110, which did not transform HEK cells, showed markedly lowered activity to direct degradation of p53. The mutants with substitutions of G for R-39, G for V-42, G for Y-43, L for F-47, and G for V-53 lost the transforming function, but they could mediate degradation of p53 at levers comparable to the activities of the wild-type and transforming mutants. Like the wild type, all of the E6 mutants were localized by immunofluorescence to the nuclei of human TS21B cells or monkey COS-I cells, except for the E6 mutant with substitution of G for Y-43 whose expression was undetectable. The levels of E6 mutants metabolically labeled in COS-l cells were comparable to those of the transforming E6s. The data indicate that EG-directed degradation of p53 is necessary but not sufficient for HPV 18-mediated transformation of HEK cells. (C) 1995 Academic Press, Inc.